GTS-21, a selective alpha7 nicotinic acetylcholine receptor agonist, ameliorates diabetic nephropathy in Leprdb/db mice

Diabetic nephropathy (DN) is a serious complicating factor in human type 2 diabetes mellitus (T2DM), and it commonly results in end-stage renal disease (ESRD) that requires kidney dialysis. Here, we report that the α7 nicotinic acetylcholine receptor (α7nAChR) agonist GTS-21 exerts a novel anti-inflammatory action to ameliorate DN, as studied using an inbred strain of Leprdb/db mice in which hyperglycemia and obesity co-exist owing to defective leptin receptor (Lepr) signaling. For this analysis, GTS-21 was administered to 10–12 week-old male and female mice as a 4 mg/kg intraperitoneal injection, twice-a-day, for 8 weeks. Kidney function and injury owing to DN were monitored by determination of plasma levels of BUN, creatinine, KIM-1 and NGAL. Histologic analysis of glomerular hypertrophy and mesangial matrix expansion were also used to assess DN in these mice. Concurrently, renal inflammation was assessed by measuring IL-6 and HMGB1, while also quantifying renal cell apoptosis, and apoptotic signaling pathways. We found that Leprdb/db mice exhibited increased markers of BUN, creatinine, NGAL, KIM-1, IL-6, cytochrome C, and HMGB-1. These abnormalities were also accompanied by histologic kidney injury (mesangial matrix expansion and apoptosis). Remarkably, all such pathologies were significantly reduced by GTS-21. Collectively, our results provide new evidence that the α7nAChR agonist GTS-21 has the ability to attenuate diabetes-induced kidney injury. Additional studies are warranted to further investigate the involvement of the vagal cholinergic anti-inflammatory reflex pathway (CAP) in ameliorating diabetic nephropathy.


Results
Actions of GTS-21 on renal function, injury and inflammation. BUN and creatinine levels in the blood are common indicators of kidney function. KIM-1 and NGAL in plasma and kidney tissue are biomarkers of acute/chronic kidney injury. In these experiments, we examine the ability of GTS-21 to attenuate the effects of T2DM on renal function, injury and inflammation. The db/db mice demonstrate a significant increase in plasma BUN (Fig. 1A) and creatinine (Fig. 1B) compared with db/+ mice (P < 0.05 vs. db/+ mice). In contrast, db/db mice treated with GTS-21 demonstrate significant reductions in plasma BUN and creatinine compared with db/db controls (P < 0.05 vs. db/db vehicle). The db/db mice also demonstrate higher levels of plasma and renal injury biomarkers KIM-1 (Fig. 1C,E) and NGAL (Fig. 1D,F) compared to db/ + vehicle controls. GTS-21 significantly attenuated the increase in plasma and renal KIM-1 and NGAL observed in the db/db mice (p < 0.05, vs. db/db vehicle).
IL-6 and HMGB-1 are inflammatory cytokines. Renal and plasma levels of these inflammatory markers were measured to assess the effects of GTS-21 on inflammation in the db/db mice. As shown in Fig. 2, db/db mice demonstrated increased plasma and renal IL-6 and HMGB-1 levels compared with db/+ mice. Treatment with GTS-21 significantly attenuated the elevations in IL-6 and HMGB-1 seen in the db/db mice (P < 0.05, Fig. 2). Collectively, these results provide evidence that α7nAChR activation plays an important role in improving kidney function, modulating renal injury and regulating renal inflammation in db/db mice.
Histological assessment of diabetic nephropathy. Mesangial matrix expansion is characterized by an increase in extracellular matrix (ECM) consisting of type IV collagen, fibronectin and laminin in the db/db mouse model of DN. As a result of the increase in ECM, the glomerular mesangial matrix and the tubulointerstitial space are expanded. When the mesangium expands, it restricts and distorts the glomerular capillaries, resulting in reduced capillary filtration in the db/db model of DN. Mesangial matrix expansion was examined by PAS staining in the different groups as shown in Fig. 3A-C. The glomerular mesangial matrix area was quantified as described in Methods and is shown in Fig. 3D. As described by others 13 , the db/db mice demonstrate a significant increase in mesangial matrix area compared with the db/+ heterozygous controls (P < 0.05 vs. db/+ vehicle). Treatment of db/db mice with GTS-21 significantly reduced the glomerular mesangial matrix area (P < 0.05 vs.

GTS-21 attenuates mitochondrial dysfunction and renal apoptosis. Apoptosis or programmed
cell death is dysregulated in many diseases, including diabetes and contributes to organ dysfunction. In DN, damaged mitochondria release cytochrome C which initiates renal apoptosis. Cytochrome C protein levels in kidney were measured by Western Blot as shown in Fig. 4A. The increase in cytochrome C levels observed in kidneys from db/db mice was significantly attenuated by GTS-21. We examined renal apoptosis by measuring the relative abundance of pro-apoptotic proteins cleaved caspase-3 ( Fig. 4B) and Bax (Fig. 4C). TUNEL staining was used to identify apoptotic cells (Fig. 4D). Renal levels of Bax (Fig. 4B) and cleaved caspase-3 ( Fig. 4C) were significantly increased in kidneys from db/db mice and were attenuated by GTS-21. Consistent with this observation, the relative abundance of TUNEL-positive cells in kidneys from db/db mice were reduced by treatment with GTS-21 ( Fig. 4D,E). These findings provide evidence that GTS-21 can attenuate mitochondrial damage and apoptosis in kidneys from db/db mice.
Activation of the PI3K/AKT and p38 MAPK pathways in db/db mice. The PI3K/AKT pathway is an intracellular signaling pathway that regulates metabolism, proliferation, cell survival and growth in response to extracellular signals 14 . The p38 mitogen-activated protein kinase (p38 MAPK) pathway is activated by extracellular stress and regulates many cellular responses including cell differentiation, apoptosis and autophagy 15 .
To investigate the role of the PI3K/AKT and p38 MAPK signaling pathways in the db/db model of DN the ratio of phosphorylated to total signaling protein in kidney was measured by Western blot analysis as an indicator of signaling pathway activation. As shown in Fig. 5A and B the ratio of phosphorylated/total PI3K and AKT is significantly decreased in kidney from db/db mice (db/db vehicle vs. db/+ vehicle, P < 0.05) and attenuated by treatment with GTS-21 (db/db GTS-21 vs. db/db vehicle, P < 0.05). In contrast, the ratio of phosphorylated/total p38 (Fig. 5C) was increased in kidney from db/db mice (db/db vehicle vs. db/+ vehicle, P < 0.05) and ameliorated by GTS-21 (db/db GTS-21 vs. db/db vehicle, P < 0.05). These results provide evidence the PI3K/AKT signaling pathway is downregulated and the p38 MAPK pathway is upregulated in kidney from db/db mice. Treatment www.nature.com/scientificreports/ with GTS-21 appears to attenuate these changes in PI3K/AKT and p38 MAPK signaling in kidneys from db/db mice.

Discussion
The pathogenesis of DN is multifactorial and includes a combination of hemodynamic, metabolic and other factors. Clinically, diabetes-induced activation of the renin-angiotensin axis is managed with antihypertensive medications. Hyperglycemia and hyperlipidemia are treated with glycemic control and lipid-lowering therapies. Despite these interventions, uncontrolled inflammation, reactive oxygen species (ROS), immune cell activation, metabolic and other factors which persist can result in progressive renal injury in patients with T2DM 16,17 . The current study examines the ability of the α7nAChR agonist, GTS-21, to attenuate renal injury in the db/db mouse model. The α7nAChR mediates many of the anti-inflammatory effects of the CAP. Studies by Tanaka et al. demonstrate vagal-mediated neuroimmune circuits which confer protection from renal injury in mice 12 . Consistent with this finding, Chatterjee et al. demonstrate the presence of the α7nAChR in mouse and human kidney cells, as well as the ability of GTS-21 to attenuate lipopolysaccharide (LPS)-induced renal inflammation and injury 18,19 . GTS-21 has also been shown to attenuate cisplatin-induced acute kidney injury 19 . Two recent reviews provide evidence for an important role of the CAP in attenuating inflammation-induced renal injury 20,21 .
We believe this to be the first study examining the effects of the CAP and α7nAChR agonist GTS-21 on DN. Our key findings include: GTS-21 improves renal function (BUN, Cr), modulates inflammation (IL-6, HMGB1), reduces kidney injury (KIM-1, NGAL), reduces histologic evidence of DN (mesangial matrix expansion) and attenuates mitochondrial damage/apoptosis (cytochrome C, Bax, cleaved caspase-3) in db/db mice with DN. These findings are associated with changes in activation of the PI3k/AKT and p38 MAPK signaling pathways Our results showing elevations in cytochrome C, pro-apoptotic proteins Bax, cleaved caspase-3 and increased apoptosis in kidneys from db/db mice are consistent with this model. While GTS-21 attenuates mitochondrial injury and the pro-apoptotic environment, reducing apoptosis in kidneys from db/db mice, the relative importance of glycemic control vs. oxidative stress, or both will require further study.
The anti-inflammatory effects of GTS-21 and the CAP are also likely to be important mechanisms for the beneficial effects of GTS-21 on DN. The increased levels of IL-6 and HMGB1 observed in kidneys from db/db mice and their reduction by GTS-21 support an anti-inflammatory mechanism of action. The anti-inflammatory effects of α7nAChR agonists are supported by numerous studies, including those of Chatterjee et al. who showed GTS-21 attenuates inflammation-induced kidney injury by LPS 19 . Activation of the α7nAChR appears to inhibit inflammation by several mechanisms 10 . In one model, α7nAChR activation recruits and activates Janus    29 . Additional studies will be necessary to determine the precise mechanisms by which GTS-21 attenuates inflammation in DN. The PI3K pathway has been implicated in glucose regulation and previous work from our laboratory showed GTS-21 activates the PI3K/AKT/mTOR signaling pathway in L cells 22 . The reduction in phosphorylated to total PI3K and AKT suggests this signaling pathway is downregulated in kidneys from db/db mice. This finding is somewhat inconsistent with previous work suggesting a role for the PI3K/AKT pathway in interstitial fibrosis in DN 30,31 . However, the increase in PI3K/AKT signaling in GTS-21 treated kidneys from non-obese mice is consistent with our results in cultured L cells 32 . Differences in experimental conditions may help to explain conflicting results regarding the PI3K pathway in DN. In contrast, our results showing p38 MAPK activation in kidneys from db/db mice is consistent with work by Lim et al. showing that activation of the p38 MAPK pathway is important in the pathogenesis of DN in the db/db mouse model 33 . The ability of GTS-21 to attenuate the p38 MAPK pathway and DN are consistent with previous work showing inhibition of p38 MAPK activation by MAP kinase kinase (MKK)3 knockout mice also attenuates renal injury in db/db mice 33 .
There are several limitations to our study. First, mutations in the leptin receptor are a rare cause of obesity and T2DM in humans. Additionally, the db/db model is limited to modest albuminuria and mesangial expansion without overt glomerulosclerosis 5,34 . Therefore, our findings may not fully translate to the human condition. Another limitation is our failure to collect urine to assay for albuminuria, a classic finding in DN. Although we show GTS-21 ameliorates many changes in db/db kidneys with DN (e.g. inflammation, mesangial matrix expansion, apoptosis, p38 MAPK signaling) our experiments were not designed to show cause and effect or fully delineate the exact mechanisms for improvements in T2DM and DN. Additional studies will be necessary to fully elucidate the mechanisms by which α7nAChR agonists attenuate experimental DN in the db/db model.
Our findings provide evidence α7nAChR agonists attenuate diabetes-induced kidney injury in the db/db model and could serve as a novel approach to ameliorating the progression of DN in patients with T2DM.

Methods
Animal models. Our studies of mice were approved by the Institutional Animal Care and Use Committee of SUNY Upstate Medical University (IACUC # 423). The studies were performed in accordance with National Institutes of Health and ARRIVE guidelines for the use of laboratory animals. Heterozygous Lepr db/+ mice (C57BLKS genetic background) were purchased from The Jackson Laboratory (Bar Harbor, ME). Homozygous Lepr db/db mice were obtained by breeding heterozygous mice so that studies could be performed in which GTS-21 action was evaluated simultaneously in Lepr db/+ and Lepr db/db littermate mice. Breeding of mice was performed at an animal core facility located at SUNY Upstate Medical University. Mice were housed in a temperature-controlled room at 22 °C under pathogen-free conditions. Male and female mice of ages 10-12 weeks were used in this study. Briefly, (1) The section was deparaffinized and hydrated to water and then oxidized in 0.5% periodic acid solution for 5 min. (2) the section was rinsed in distilled water and placed Schiff reagent for 15 min, and then washed in lukewarm tap water for 5 min. (3) The section was with Counterstain in Mayer's hematoxylin for 1 min, and then washed in tap water for 5 min. (4) The section was dehydrated and covered using CYTOSEAL tm 60 (Cat. #: 8310-4, Thermo Scientific). Digital images from 20 glomeruli per animal were used at 400 × magnification under a microscope (Nikon, Melville, NY) to assess renal pathology alternations. Relative mesangial matrix area was calculated according to the method as descripted previously using Image J 13 . Relative mesangial matrix area (%) is defined as the fractional area of mesangial matrix area (PAS-positive and nuclei-free area in the mesangium) over glomerular area (along the edge of the capillary loops of glomeruli).

Western blot analysis.
Tissues were incubated with protein lysis buffer for 30 min at 4 °C, then centrifuged at 13,000 rpm for 5 min to sediment cell debris. Supernatants were collected used for Western blot. The same amount (20 µg) of proteins were loaded and were separated by SDS-PAGE, and then transferred to PVDF membrane (Cat. #: IPVH00010, Millipore Co., Ltd. USA). The membranes were blocked with 5% nonfat milk (Cat. #: MI17200, Research Products International) in Tris-buffered TBS plus 0.5% Tween-20 (Cat. #: BP337, Fisher Scientific) for 1 h at room temperature and incubated overnight at 4 °C with the indicated primary . After primary antibody incubation, the blot was incubated with HRP-conjugated secondary antibody for 1 h at room temperature. Antibody-antigen complexes were visualized with ECL (Cat. #: 34580, Thermo Scientific, IL) and analyzed quantitatively by densitometry with Image J software. The relative density of immunoreactive bands was normalized to the density of the corresponding β-actin.
Assessment of apoptotic cells. For detecting renal apoptotic cells, deoxynucleotidyl transferase mediated dUTP nick-end labeling (TUNEL)kit (Cat. #: C10617, Invitrogen, Eugene, OR) was used according to the manufacturer's instructions. Briefly, the sections were deparaffinized with xylene, dehydrated through a graded alcohol series to water, and treated with permeabilization solution. The labeling reaction was performed using a solution containing terminal deoxynucleotidyl transferase. After staining, the sections were mounted by fluoroshield mounting medium with DAPI (Cat. #: ab104139, Abcam Inc, Cambridge, MA) to visualize nuclei. Apoptotic cells were quantified by counting TUNEL positive cells from five randomly selected consecutive fields at 400 magnification by two experienced investigators in a blinded manner. Apoptotic index was calculated as the number of TUNEL positive cells expressed as percentage of total cells.
Statistical analysis. The data are expressed as the mean ± SEM (with Gaussian distribution) or median ± interquartile range (with no Gaussian distribution) analyzed using GraphPad Prism 9. The Mann-Whitney test or t test were used to compare differences between two independent groups. One-way analysis of variance (one-way ANOVA) with Bonferroni's multiple comparisons test or Kruskal-Wallis test with Dunn's multiple comparison test were used to determine multiple group differences. P values of < 0.05 were considered significant. Data was obtained from three or more independent experiments.

Data availability
Datasets used in this study are available from the corresponding authors upon reasonable request.